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. 2018 Apr 21;10(4):126. doi: 10.3390/cancers10040126

Figure 2.

Figure 2

Influence of oxygen levels and treatment on apoptosis of NCI-H2228 cells. Cells were treated for 72 h (A–E) or 24 h (F) with vehicle, CDDP, APR-246, or CDDP/APR-246. (A) Apoptosis was monitored using the IncuCyte Annexin V Green reagent under normoxic conditions. Presented as mean ± SD Green Object Confluence/Phase Object Confluence of 3 replicates. (B) Viability was assessed by flow cytometry using the AnnexinV/PI assay. AnnV-/PI-cells are marked as viable cells. Data is presented as mean ± SD of 3 independent experiments. (C,D) Apoptosis/cell death was assessed by flow cytometry under normoxic and hypoxic conditions. Percentage of AnnV−/PI−; AnnV+/PI−; AnnV+/PI+ and AnnV−/PI+ cells is shown as mean ± SD of 3 independent experiments. * p < 0.05 compared to vehicle treated sample; ** p < 0.05 compared to vehicle, CDDP and APR-246 treated samples. Dotplots are presented in Figure S1. (E) Caspase activity was monitored using the IncuCyte Caspase-3/7 Green reagent. Presented as mean ± SD of 3 replicates Green Object Confluence/Phase Object Confluence. (F) Procaspase-3 and cleaved caspase-3 levels were determined by Western blotting; β-actin was used as an internal standard. ARD: Adjusted Relative Density; Nx: normoxia; Hx: hypoxia.